Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered question. Indeed, proteases are regulated by a complex series of post- translational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes. To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody- based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.